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Cortical hyperexcitability and mislocalization of the RNA-binding protein TDP43 are highly conserved features in amyotrophic lateral sclerosis (ALS). Nevertheless, the relationship between these phenomena remains poorly defined. Here, we showed that hyperexcitability recapitulates TDP43 pathology by upregulating shortened TDP43 (sTDP43) splice isoforms. These truncated isoforms accumulated in the cytoplasm and formed insoluble inclusions that sequestered full-length TDP43 via preserved N-terminal interactions. Consistent with these findings, sTDP43 overexpression was toxic to mammalian neurons, suggesting neurodegeneration arising from complementary gain- and loss-of-function mechanisms. In humans and mice, sTDP43 transcripts were enriched in vulnerable motor neurons, and we observed a striking accumulation of sTDP43 within neurons and glia of ALS patients. Collectively, these studies uncover a pathogenic role for alternative TDP43 isoforms in ALS, and implicate sTDP43 as a key contributor to the susceptibility of motor neurons in this disorder.

Intron retention has long been an exemplar of regulated splicing with case studies of individual events serving as models that provided key mechanistic insights into the process of splicing control. In organisms such as plants and budding yeast, intron retention is well understood as a major mechanism of gene expression regulation. In contrast, in mammalian systems, the extent and functional significance of intron retention have, until recently, remained greatly underappreciated. Technical challenges to the global detection and quantitation of transcripts with retained introns have often led to intron retention being overlooked or dismissed as “noise”. Now, however, with the wealth of information available from high-throughput deep sequencing, combined with focused computational and statistical analyses, we are able to distinguish clear intron retention patterns in various physiological and pathological contexts. Several recent studies have demonstrated intron retention as a central component of gene expression programs during normal development as well as in response to stress and disease. Furthermore, these studies revealed various ways in which intron retention regulates protein isoform production, RNA stability and translation efficiency, and rapid induction of expression via post-transcriptional splicing of retained introns. In this review, we highlight critical findings from these transcriptomic studies and discuss commonalties in the patterns prevalent in intron retention networks at the functional and regulatory levels.

The constitutively active androgen receptor (AR) splice variant 7 (AR-V7) plays an important role in the progression of castration-resistant prostate cancer (CRPC). Although biomarker studies established the role of AR-V7 in resistance to AR-targeting therapies, how AR-V7 mediates genomic functions in CRPC remains largely unknown. Using a ChIP-exo approach, we show AR-V7 binds to distinct genomic regions and recognizes a full-length androgen-responsive element in CRPC cells and patient tissues. Remarkably, we find dramatic differences in AR-V7 cistromes across diverse CRPC cells and patient tissues, regulating different target gene sets involved in CRPC progression. Surprisingly, we discover that HoxB13 is universally required for and colocalizes with AR-V7 binding to open chromatin across CRPC genomes. HoxB13 pioneers AR-V7 binding through direct physical interaction, and collaborates with AR-V7 to up-regulate target oncogenes. Transcriptional coregulation by HoxB13 and AR-V7 was further supported by their coexpression in tumors and circulating tumor cells from CRPC patients. Importantly, HoxB13 silencing significantly decreases CRPC growth through inhibition of AR-V7 oncogenic function. These results identify HoxB13 as a pivotal upstream regulator of AR-V7–driven transcriptomes that are often cell context-dependent in CRPC, suggesting that HoxB13 may serve as a therapeutic target for AR-V7–driven prostate tumors.

Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) cause hearing loss without vestibular dysfunction in both mice and humans, we investigated the contribution of Tmc1 and the closely related Tmc2 to mechanotransduction in mice. We found that Tmc1 and Tmc2 were expressed in mouse vestibular and cochlear hair cells and that GFP-tagged TMC proteins localized near stereocilia tips. Tmc2 expression was transient in early postnatal mouse cochlear hair cells but persisted in vestibular hair cells. While mice with a targeted deletion of Tmc1 (Tmc1(Δ) mice) were deaf and those with a deletion of Tmc2 (Tmc2(Δ) mice) were phenotypically normal, Tmc1(Δ)Tmc2(Δ) mice had profound vestibular dysfunction, deafness, and structurally normal hair cells that lacked all mechanotransduction activity. Expression of either exogenous TMC1 or TMC2 rescued mechanotransduction in Tmc1(Δ)Tmc2(Δ) mutant hair cells. Our results indicate that TMC1 and TMC2 are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex. Our data also suggest that persistent TMC2 expression in vestibular hair cells may preserve vestibular function in humans with hearing loss caused by TMC1 mutations.

Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. To produce a protein, a gene’s DNA is first copied to make molecules of messenger RNA (mRNA). The mRNAs pass through a molecular machine known as the ribosome, which translates the genetic code to make a protein. Not all of an mRNA is translated to make a protein; the “untranslated” regions play crucial roles in regulating how much of the protein is produced. In animals, plants and other eukaryotes, many mRNAs are made up of small pieces that are “spliced” together. During this process, proteins are deposited on the mRNA to mark the splice junctions, which are then cleared when the mRNA is translated. Many different mRNAs can be produced from the same gene by splicing different combinations of RNA pieces. Each of these mRNA “isoforms” can, in principle, contain a unique set of features that control its translation. Hence each mRNA isoform can be translated differently so that different amounts of the corresponding protein product are produced. However, the relationship between the variety of isoforms and the control of translation is complex and not well understood. To address these questions, Floor and Doudna measured the translation of over 60,000 mRNA isoforms made from almost 14,000 human genes. The experiments show that untranslated regions at the end of the mRNA (known as the 3′ end) strongly influence translation, even if the protein coding regions remain the same. Furthermore, the data showed that mRNAs with more splice junctions are translated better, implying an mRNA has some sort of memory of how many junctions it had even after the protein markers have been cleared. Next, Floor and Doudna inserted regulatory sequences from differently translated isoforms into an unrelated “reporter” gene. This dramatically changed the amount of protein produced from the reporter gene, in a manner predicted by the earlier experiments. Untranslated regions at the beginning of the mRNAs (known as the 5′ end) controlled the amount of protein produced from the reporter consistently across different types of cells from the body. On the other hand, the 3′ regions can tune the level of protein production in particular types of cells. Floor and Doudna’s findings demonstrate that differences between mRNA isoforms of a gene can have a big effect on the level of protein production. Changes in the types of mRNA made from a gene are often associated with human diseases, and these findings suggest one reason why. Additionally, the ability to engineer translation of an mRNA using the data is likely to aid the development of mRNA-based therapies.

Hormone-induced brain masculinization occurs during a perinatal sensitive period but endures into adulthood. Researchers explored DNA methylation as a candidate mechanism. Methylation is higher in female brain and suppresses masculinization genes, which are liberated by hormone-induced reductions in DNMT activity in males. Pharmacological inhibition of DNMTs reduces methylation, masculinizes female brain and behavior and reopens the sensitive period. The developing mammalian brain is destined for a female phenotype unless exposed to gonadal hormones during a perinatal sensitive period. It has been assumed that the undifferentiated brain is masculinized by direct induction of transcription by ligand-activated nuclear steroid receptors. We found that a primary effect of gonadal steroids in the highly sexually dimorphic preoptic area (POA) is to reduce activity of DNA methyltransferase (Dnmt) enzymes, thereby decreasing DNA methylation and releasing masculinizing genes from epigenetic repression. Pharmacological inhibition of Dnmts mimicked gonadal steroids, resulting in masculinized neuronal markers and male sexual behavior in female rats. Conditional knockout of the de novo Dnmt isoform, Dnmt3a, also masculinized sexual behavior in female mice. RNA sequencing revealed gene and isoform variants modulated by methylation that may underlie the divergent reproductive behaviors of males versus females. Our data show that brain feminization is maintained by the active suppression of masculinization via DNA methylation.

Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host 1 . Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility 2 , 3 . Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei , using long-read sequencing technology and conserved features of chromosome folding 4 . Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation. Long-read sequencing allows the assembly of antigen-gene arrays in Trypanosoma brucei and, coupled with deletion experiments, demonstrates that histone variants act as a molecular link between genome architecture, chromatin conformation and antigen variation.

Insects are the only known animals in which sexual differentiation is controlled by sex-specific splicing. The doublesex transcription factor produces distinct male and female isoforms, which are both essential for sex-specific development. dsx splicing depends on transformer, which is also alternatively spliced such that functional Tra is only present in females. This pathway has evolved from an ancestral mechanism where dsx was independent of tra and expressed and required only in males. To reconstruct this transition, we examined three basal, hemimetabolous insect orders: Hemiptera, Phthiraptera, and Blattodea. We show that tra and dsx have distinct functions in these insects, reflecting different stages in the changeover from a transcription-based to a splicing-based mode of sexual differentiation. We propose that the canonical insect tra-dsx pathway evolved via merger between expanding dsx function (from males to both sexes) and narrowing tra function (from a general splicing factor to dedicated regulator of dsx).

Treatment of locally advanced and metastatic prostate cancer (PC) with androgen receptor–targeting (AR-targeting) therapies has limited durability, with disease eventually progressing to castrate-resistant PC (CRPC). Constitutively active AR splice variants (AR-Vs), such as AR-V7, play a key role in driving treatment resistance and disease progression. Importantly, the failure to attenuate AR-V function represents a major unmet clinical need, and as such, defining how AR-Vs are generated is likely to yield new therapeutic targets. Our knowledge of factors that mediate splicing of AR-V–encoding mRNAs remains limited. Here, we have employed an RNA-targeting CasRx approach to identify selective protein interactors of AR-V7 mRNA in PC. TRA2B and its ortholog, TRA2A, were identified as splicing regulators of AR transcripts that facilitate AR-V synthesis at the expense of full-length AR isoforms. TRA2B expression correlated with AR-V7 transcript in CRPC and attenuation of TRA2-mediated splicing diminished PC cell growth. Exploiting TRA2B function may therefore provide new therapeutic opportunities in advanced disease.

New androgen receptor (AR) pathway inhibitors (ARPIs) in clinical development, including AR degraders and CYP11A inhibitors, largely target ligand-dependent AR activation and have reported antitumor activity in metastatic castration-resistant prostate cancer (mCRPC) resistant to established ARPIs, predominately against tumors with AR mutations. We hypothesized that AR -mutated mCRPC exhibits lower AR splice variant 7 (AR-V7) expression and remains full-length–AR (FL-AR) driven, explaining, in part, the antitumor activity of these AR ligand–binding domain (LBD) targeting drugs. The data herein demonstrate that mCRPC tissue biopsies with detectable AR mutations express significantly lower levels of AR-V7 protein and associate with better overall survival and enhanced sensitivity to ARPIs. This is independent of differences in the total number of global splicing events but may be related to differences in splicing factor expression between AR -mutated and nonmutated mCRPC. In conclusion, AR -mutated mCRPC frequently exhibits low AR-V7 expression, arguably explaining the enhanced sensitivity to ARPIs observed in these cancers. Consequently, AR mutation status may serve as a biomarker to predict response to AR-directed therapies.